(1) Field of the Invention
The present invention relates generally to microorganism binding with a substrate bound capture agent having a saccharide moiety and a lectin. In particular, the present invention relates to a method of binding a microorganism on the substrate using the capture agent and lectin in an assay method and test kit.
(2) Description of the Related Art
Rapid methods for bacterial detection are essential in food, industrial, environmental monitoring, clinical diagnostics and biodefense to allow faster decisions to be made with respect to food poisoning, water contamination, the presence of disease and, therefore, treatment options. Most conventional methods (e.g. plating and culturing, biochemical tests, microscopy, flow cytometry, luminescence) are time consuming, often requiring 1-2 days to obtain results. Although much faster detection methods such as immunosensors or DNA chips are becoming available, they have failed to gain wide acceptance due to the high user expertise required, high cost of labeling reagents, and low stability of antibody and DNA recognition elements. As a result, a rapid, quantitative, sensitive and specific method for one step bacterial detection is highly sought after.
Cell surface carbohydrates (glycans) and adhesin molecules are major components of the outer surface of cells and are often characteristic of the cell types. Many adhesin molecules are lectins that have carbohydrate binding activities. Glycans and adhesins are the first interface to the biotic and abiotic environment of the cell. The interactions of glycans with carbohydrate binding proteins (lectins) are perhaps the most significant and fundamental molecular recognition events in biological systems including bacterial pathogenesis, tumor cell metastasis, and inflammation. To understand the biological roles of a particular carbohydrate or to evaluate a lectin adhesin as a disease biomarker, one must to determine when, where and how much a carbohydrate and/or lectin adhesin is expressed. The expression of carbohydrate structures changes dramatically during cell development and the carbohydrates from different organisms display tremendous variations in structure and function. A similar situation exists for bacterial cell surface lectin adhesin expression. As a result, the carbohydrate and/or lectin adhesin expression levels are extremely difficult to measure and present a formidable challenge for studying and characterizing their roles in cell biology.
In recent years, advances in the fields of combinatorial carbohydrate synthesis and automated carbohydrate synthesis have made available a great number of glycans for study. (32Jelinek, R.; Kolusheva, S. “Carbohydrate Biosensors.” Chem. Rev. 2004, 104, 5987-6015) Carbohydrate microarrays were developed to study the carbohydrate-cell interaction and to detect pathogens.(14)-(16) Carbohydrate-arrays allow for the analysis of protein carbohydrate interactions in a variety of glycobiology systems, but they do not allow the direct examination of changes in glycosylation. Therefore, the bound lectin arrays that allow quick assess of bacterial cell surface carbohydrate compositions were developed. Unfortunately similar to many protein arrays, they suffer some loss of binding activity in the coupling of lectin to the arrays. The large sizes of lectins also increase their susceptibility to proteases and encourage non-specific binding. Most previously reported carbohydrate and lectin arrays are one dimensional and use fluorescence label for detection. Fluorescence labeling of the bacteria cells requires additional steps. The presence of the labels themselves can introduce additional interferences to the “true” binding process. Fluorescent detection can also suffer from high background fluorescence which may produce false positive results. U.S. Patent Application Publication No. 2006/0014232 to Inagawa et al. teaches immobilization of biomolecules, provided with at least one tag, to a substrate. The substrate has binding sites for the tags and activated reactive groups capable of forming covalent bonds with the biomolecules. The biomolecules can be immobilized to prepare a sensor chip used for surface plasmon resonance or quartz-crystal microbalance techniques. The prior art does not teach the use of lectins to bind a microorganism to the substrate.
While the related art teach bacterial detection methods, there still exists a need for rapid, quantitative, sensitive and specific microorganisms analysis and detection method and test kit. See references: ((17Nangia-Makker, P.; Conklin, J. Hogan, V.; Raz, A. “Carbohydrate-binding proteins in cancer, and their ligands as therapeutic agents.” Trends in Molecular Medicine 2002, 8, 187-192.) (18Stevenson, G.; Neal, B.; Liu, D.; Hobbs, M.; Packer, N. H.; Batley, M.; Redmond, J. W.; Lindquist, L.; Reeves, P. “Structure of the O-Antigen of Escherichia-Coli-K-12 and the Sequence of Its Rfb Gene-Cluster.” Journal of Bacteriology 1994, 176, 4144-4156); (19Lee, Y. C.; Lee, R. T. “Carbohydrate-Protein Interactions: Basis of Glycobiology.” Accounts of Chemical Research 1995, 28, 321-7); (21Williams and Davies, Trends in biotechnology 2001, 19, 356-62); (22Lindhorst, T. K. “Artificial multivalent sugar ligands to understand and manipulate carbohydrate-protein interactions.” Topics in Current Chemistry 2002, 218, 201-235); (23Houseman, B. T.; Mrksich, M. “Model systems for studying polyvalent carbohydrate binding interactions.” Topics in Current Chemistry 2002, 218, 1-44); (24Liang, R.; Loebach, J.; Horan, N.; Ge, M.; Thompson, C.; Yan, L.; Kahne, D. “Polyvalent binding to carbohydrates immobilized on an insoluble resin.” Proceedings of the National Academy of Sciences of the United States of America 1997, 94, 10554-10559); (25Mathai Mammen, S.-K. C. G. M. W. “Polyvalent Interactions in Biological Systems: Implications for Design and Use of Multivalent Ligands and Inhibitors.” Angewandte Chemie International Edition 1998, 37, 2754-2794); (26Shinohara, Y.; Hasegawa, Y.; Kaku, H.; Shibuya, N. “Elucidation of the mechanism enhancing the avidity of lectin with oligosaccharides on the solid phase surface.” Glycobiology 1997, 7, 1201-1208); (27Kolb, H. C.; Finn, M. G.; Sharpless, K. B. “Click Chemistry: Diverse chemical function from a few good reactions.” Angewandte Chemie-International Edition 2001, 40, 2004-+); (28Ratner, D. M.; Adams, E. W.; Disney, M. D.; Seeberger, P. H. “Tools for glycomics: Mapping interactions of carbohydrates in biological systems.” Chembiochem 2004, 5, 1375-1383); (29Mann, D. A.; Kanai, M.; Maly, D. J.; Kiessling, L. L. “Probing Low Affinity and Multivalent Interactions with Surface Plasmon Resonance: Ligands for Concanavalin A.” Journal of the American Chemical Society 1998, 120, 10575-10582); (30Ostuni, E.; Chapman, R. G.; Liang, M. N.; Meluleni, G.; Pier, G.; Ingber, D. E.; Whitesides, G. M. “Self-assembled monolayers that resist the adsorption of proteins and the adhesion of bacterial and mammalian cells.” Langmuir 2001, 17, 6336-6343); (31Fung, Y. S.; Wong, Y. Y. “Self-assembled monolayers as the coating in a quartz piezoelectric crystal immunosensor to detect Salmonella in aqueous solution.” Analytical Chemistry 2001, 73, 5302-5309); (33Poxton, I. R. “Antibodies to lipopolysaccharide.” Journal of Immunological Methods 1995, 186, 1-15) and (34Feizi, T.; Fazio, F.; Chai, W.; Wong Chi, H. “Carbohydrate microarrays—a new set of technologies at the frontiers of glycomics.” Current opinion in structural biology 2003, 13, 637-45).